Seminal fluid proteins (Sfps) are an important, yet often overlooked, component of fertility in animals from insects to mammals. Sfps have significant effects on the reproductive physiology of mated females, on storage and release of sperm inside females and, as recently shown in mice, on the phenotype of progeny. Male animals abnormal for specific Sfps are sterile or subfertile; evidence for this extends to humans. Some Sfps bind tightly to sperm; others remain in the seminal plasma and therefore are removed during some Assisted Reproductive Technologies (ARTs). Yet despite their importance in reproduction, little is known about exactly how Sfps act to influence the female. This proposal combines genetic, physiological, evolutionary, and protein methodologies to address two pressing questions about Sfps' action: (1) How, precisely, do Sfps interact with the female to cause post- mating effects? (2) How do Sfps bind tightly to sperm and exert effects on sperm storage? We address these by using the experimental strengths of Drosophila, which we have established as a premier genetic model system with which to dissect Sfp function and whose Sfps have many molecular and phenomenological parallels to those of mammals. To address the first question, Aim 1 focuses on ovulin, a Drosophila Sfp that stimulates ovulation. Ovulin induces octopaminergic signaling, which in turn regulates muscle contraction in the reproductive tract, causing an increase in ovulation rate. We will identify the female's receptor for ovulin (OvR) by testing our candidate GPCRs for ovulation phenotypes upon knockdown, and for binding to ovulin in cultured cells. Then, using targeted knockdowns of OvR we will test the hypothesis that ovulin acts directly on the nervous system. We also will determine whether the proteolytic processing of ovulin that occurs when it enters the female enhances ovulin's activity and its binding to OvR. To address the mechanism and function of sperm-bound Sfps, Aim 2 focuses on the Drosophila sex peptide, SP. We recently defined a network of Sfps that binds SP to sperm. We will test whether male contributions are sufficient for SP's sperm binding, or whether females also contribute factors, by determining whether SP can bind to sperm in pure ejaculates. We will then identify the sperm protein that is required for SP binding. SP action is required to efficiently release sperm from storage. In the final part of Aim 2 we will test the hypothesis that SP does so through the nervous system (and octopaminergic signaling) rather than by action within the sperm storage organs. Elucidating how Sfps interact with and affect the female at the molecular level is a very new area that is only now becoming amenable to study. Results of the proposed work are relevant to understanding and diagnosing Sfp-based infertilities and in considering strategies for ARTs, which may benefit from inclusion of critical Sfps. Additionally, understanding reproductive molecules in Dipteran insects will assist in strategies to control mosquitoes that transmit viruses that cause serious human diseases like dengue and malaria.